Hereditary stomatocytosis (HSt) is a rare, autosomal dominant, congenital hemolytic anemia distinguished by a "mouth-shaped" ("stomatocytic") red cell morphology, a marked leak to the univalent cations Na+ and K+, and the absence on SDS-PAGE of band 7.2. This protein has been recently cloned, is of 31,700 MW, and has been named stomatin in recognition of its absence in HSt patients. It has structural features of a transmembrane protein, with a (predicted) single membrane spanning segment and a large cytoplasmic domain. Several lines of evidence indicate that this protein by itself does not form an ion transporter, and preliminary data (surprisingly) indicate that the coding region of this gene is normal in HSt patients. Their reticulocyte stomatin mRNA levels also approximate those of normal individuals. Since this is a dominant disorder, these results suggest that extrinsic factors may act to stabilize stomatin. We postulate that stomatin binds to elements of the cytoskeleton, and also functions as a trans-acting regulator of K+ ion conductance by forming a "plug" or "gate" controlling the flow of ions through an as yet unidentified channel. The overall goals of this project are thus to identify i) the proteins in normal erythrocytes that interact with stomatin; ii) the functional domains of stomatin; and iii) the status of these interacting proteins in patients with hereditary stomatocytosis. These goals will be approached by using in vitro assays to assess the capacity of recombinant stomatin to bind other known red cell membrane proteins; chemical cross-linking, expression cloning, and genetic selection assays to identify potential unknown interacting proteins; and deletional mutagenesis of stomatin to assess which domains are responsible for its membrane skeletal protein interactions, and which domains suppress the monovalent cation leak in HSt patients. The long-term goal of these studies is to identify the molecular basis of hereditary stomatocytosis, and to define novel protein interactions that contribute to erythrocyte membrane structure and function. If it is found that stomatin acts in trans-fashion to block a monovalent cation channel, as is hypothesized, these studies will also establish a new paradigm for the control of ion- transport that is similar but complementary to the cis-acting mechanisms proposed for the voltage-gated Shaker channel of Drosophila and the thrombin receptor of platelets.